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A nuclear export signal in Kap95p is required for both recycling the import factor and interaction with the nucleoporin GLFG repeat regions of Nup116p and Nup100p.

Iovine MK, Wente SR - J. Cell Biol. (1997)

Bottom Line: Mutation of the NES in Kap95p resulted in a temperaturesensitive import mutant, and immunofluorescence microscopy experiments showed that the mutated Kap95p was not recycled but instead localized in the nucleus and at the nuclear envelope.The protein A-tagged Nup116p complex also specifically contained Gle2p.These results support a model in which a step in the recycling of Kap95p is mediated by interaction of an NES with GLFG regions.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
During nuclear import, cytosolic transport factors move through the nuclear pore complex (NPC) to the nuclear compartment. Kap95p is required during import for docking the nuclear localization signal-receptor and ligand to the NPC. Recycling of this factor back to the cytoplasm is necessary for continued rounds of import; however, the mechanism for Kap95p recycling is unknown. We have determined that recycling of Kap95p requires a nuclear export signal (NES). A region containing the NES in Kap95p was sufficient to mediate active nuclear export in a microinjection assay. Moreover, the NES was necessary for function. Mutation of the NES in Kap95p resulted in a temperaturesensitive import mutant, and immunofluorescence microscopy experiments showed that the mutated Kap95p was not recycled but instead localized in the nucleus and at the nuclear envelope. Srp1p, the yeast nuclear localization signal-receptor, also accumulated in the nuclei of the arrested kap95 mutant cells. Wild-type and NES-mutated Kap95p both bound Gsp1p (the yeast Ran/TC4 homologue), Srp1p, and the FXFG repeat region of the nucleoporin Nup1p. In contrast, the NES mutation abolished Kap95p interaction with the GLFG repeat regions from the nucleoporins Nup116p and Nup100p. In vivo interaction was demonstrated by isolation of Kap95p from yeast nuclear lysates in either protein A-tagged Nup116p or protein A-tagged Nup100p complexes. The protein A-tagged Nup116p complex also specifically contained Gle2p. These results support a model in which a step in the recycling of Kap95p is mediated by interaction of an NES with GLFG regions. Analysis of genetic interactions suggests Nup116p has a primary role in Kap95p recycling, with Nup100p compensating in the absence of Nup116p. This finding highlights an important role for a subfamily of GLFG nucleoporins in nuclear export processes.

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Nuclear import capacity is diminished in arrested  kap95-L63A cells. (A) To assay nuclear import, wild-type  (SWY1312) and kap95-L63A (SWY1313) strains were transformed with the plasmid expressing NLS–β-galactosidase under  GAL10 (pNLS-E1). Expression was induced by the addition of  2% galactose, and the cells were shifted to growth at 37°C. After  3 h at 37°C, the cells were fixed and processed for immunofluorescence microscopy. Localization of the reporter was determined using mAbs against β-galactosidase. (B) Export of  poly(A)+ RNA was not inhibited. Wild-type and kap95-L63A  cells grown in YEPD at 23°C were shifted to growth at 37°C and  processed for in situ hybridization with a digoxigenin-oligo- (dT)30 probe. FITC-conjugated anti-digoxigenin antibodies were  used to localize probe binding. Exposure and printing times are  identical for wild-type and mutant cells in the given experiment.  Coincident DAPI staining is shown. Bar, 5 μm.
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Figure 6: Nuclear import capacity is diminished in arrested kap95-L63A cells. (A) To assay nuclear import, wild-type (SWY1312) and kap95-L63A (SWY1313) strains were transformed with the plasmid expressing NLS–β-galactosidase under GAL10 (pNLS-E1). Expression was induced by the addition of 2% galactose, and the cells were shifted to growth at 37°C. After 3 h at 37°C, the cells were fixed and processed for immunofluorescence microscopy. Localization of the reporter was determined using mAbs against β-galactosidase. (B) Export of poly(A)+ RNA was not inhibited. Wild-type and kap95-L63A cells grown in YEPD at 23°C were shifted to growth at 37°C and processed for in situ hybridization with a digoxigenin-oligo- (dT)30 probe. FITC-conjugated anti-digoxigenin antibodies were used to localize probe binding. Exposure and printing times are identical for wild-type and mutant cells in the given experiment. Coincident DAPI staining is shown. Bar, 5 μm.

Mentions: Since Kap95p is an essential import factor, mutations that perturb its function should coincidentally alter nuclear protein import capacity. In particular, if Kap95p was not recycled to the cytoplasm, the import capacity of the cell may become noticeably diminished with only limited (newly synthesized) Kap95p available in the cytoplasm. To examine the effect of the temperature-sensitive kap95L63A allele on import, the kap95-L63A strain was transformed with a plasmid expressing an NLS–β-galactosidase fusion protein under control of the inducible GAL10 promoter (Underwood and Fried, 1990). If import is diminished during growth at the nonpermissive temperature (37°C), the NLS–β-galactosidase will not accumulate in the nucleus. To induce reporter protein synthesis, cells grown in raffinose-containing media at 23°C were shifted to galactose-containing media and growth at 37°C. Localization of the fusion protein was determined by indirect immunofluorescence microscopy with anti–β-galactosidase antibodies. When grown at 37°C, the wild-type cells efficiently imported the NLS–β-galactosidase as reflected by the bright nuclear staining (Fig. 6 A). In contrast, the kap95-L63A mutant cells at 37°C showed diminished nuclear staining and the cytoplasmic signal was significantly enhanced (Fig. 6 A). Therefore, the NES-mutated protein was not capable of sustaining wild-type levels of nuclear import capacity at the nonpermissive temperature.


A nuclear export signal in Kap95p is required for both recycling the import factor and interaction with the nucleoporin GLFG repeat regions of Nup116p and Nup100p.

Iovine MK, Wente SR - J. Cell Biol. (1997)

Nuclear import capacity is diminished in arrested  kap95-L63A cells. (A) To assay nuclear import, wild-type  (SWY1312) and kap95-L63A (SWY1313) strains were transformed with the plasmid expressing NLS–β-galactosidase under  GAL10 (pNLS-E1). Expression was induced by the addition of  2% galactose, and the cells were shifted to growth at 37°C. After  3 h at 37°C, the cells were fixed and processed for immunofluorescence microscopy. Localization of the reporter was determined using mAbs against β-galactosidase. (B) Export of  poly(A)+ RNA was not inhibited. Wild-type and kap95-L63A  cells grown in YEPD at 23°C were shifted to growth at 37°C and  processed for in situ hybridization with a digoxigenin-oligo- (dT)30 probe. FITC-conjugated anti-digoxigenin antibodies were  used to localize probe binding. Exposure and printing times are  identical for wild-type and mutant cells in the given experiment.  Coincident DAPI staining is shown. Bar, 5 μm.
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Related In: Results  -  Collection

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Figure 6: Nuclear import capacity is diminished in arrested kap95-L63A cells. (A) To assay nuclear import, wild-type (SWY1312) and kap95-L63A (SWY1313) strains were transformed with the plasmid expressing NLS–β-galactosidase under GAL10 (pNLS-E1). Expression was induced by the addition of 2% galactose, and the cells were shifted to growth at 37°C. After 3 h at 37°C, the cells were fixed and processed for immunofluorescence microscopy. Localization of the reporter was determined using mAbs against β-galactosidase. (B) Export of poly(A)+ RNA was not inhibited. Wild-type and kap95-L63A cells grown in YEPD at 23°C were shifted to growth at 37°C and processed for in situ hybridization with a digoxigenin-oligo- (dT)30 probe. FITC-conjugated anti-digoxigenin antibodies were used to localize probe binding. Exposure and printing times are identical for wild-type and mutant cells in the given experiment. Coincident DAPI staining is shown. Bar, 5 μm.
Mentions: Since Kap95p is an essential import factor, mutations that perturb its function should coincidentally alter nuclear protein import capacity. In particular, if Kap95p was not recycled to the cytoplasm, the import capacity of the cell may become noticeably diminished with only limited (newly synthesized) Kap95p available in the cytoplasm. To examine the effect of the temperature-sensitive kap95L63A allele on import, the kap95-L63A strain was transformed with a plasmid expressing an NLS–β-galactosidase fusion protein under control of the inducible GAL10 promoter (Underwood and Fried, 1990). If import is diminished during growth at the nonpermissive temperature (37°C), the NLS–β-galactosidase will not accumulate in the nucleus. To induce reporter protein synthesis, cells grown in raffinose-containing media at 23°C were shifted to galactose-containing media and growth at 37°C. Localization of the fusion protein was determined by indirect immunofluorescence microscopy with anti–β-galactosidase antibodies. When grown at 37°C, the wild-type cells efficiently imported the NLS–β-galactosidase as reflected by the bright nuclear staining (Fig. 6 A). In contrast, the kap95-L63A mutant cells at 37°C showed diminished nuclear staining and the cytoplasmic signal was significantly enhanced (Fig. 6 A). Therefore, the NES-mutated protein was not capable of sustaining wild-type levels of nuclear import capacity at the nonpermissive temperature.

Bottom Line: Mutation of the NES in Kap95p resulted in a temperaturesensitive import mutant, and immunofluorescence microscopy experiments showed that the mutated Kap95p was not recycled but instead localized in the nucleus and at the nuclear envelope.The protein A-tagged Nup116p complex also specifically contained Gle2p.These results support a model in which a step in the recycling of Kap95p is mediated by interaction of an NES with GLFG regions.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
During nuclear import, cytosolic transport factors move through the nuclear pore complex (NPC) to the nuclear compartment. Kap95p is required during import for docking the nuclear localization signal-receptor and ligand to the NPC. Recycling of this factor back to the cytoplasm is necessary for continued rounds of import; however, the mechanism for Kap95p recycling is unknown. We have determined that recycling of Kap95p requires a nuclear export signal (NES). A region containing the NES in Kap95p was sufficient to mediate active nuclear export in a microinjection assay. Moreover, the NES was necessary for function. Mutation of the NES in Kap95p resulted in a temperaturesensitive import mutant, and immunofluorescence microscopy experiments showed that the mutated Kap95p was not recycled but instead localized in the nucleus and at the nuclear envelope. Srp1p, the yeast nuclear localization signal-receptor, also accumulated in the nuclei of the arrested kap95 mutant cells. Wild-type and NES-mutated Kap95p both bound Gsp1p (the yeast Ran/TC4 homologue), Srp1p, and the FXFG repeat region of the nucleoporin Nup1p. In contrast, the NES mutation abolished Kap95p interaction with the GLFG repeat regions from the nucleoporins Nup116p and Nup100p. In vivo interaction was demonstrated by isolation of Kap95p from yeast nuclear lysates in either protein A-tagged Nup116p or protein A-tagged Nup100p complexes. The protein A-tagged Nup116p complex also specifically contained Gle2p. These results support a model in which a step in the recycling of Kap95p is mediated by interaction of an NES with GLFG regions. Analysis of genetic interactions suggests Nup116p has a primary role in Kap95p recycling, with Nup100p compensating in the absence of Nup116p. This finding highlights an important role for a subfamily of GLFG nucleoporins in nuclear export processes.

Show MeSH
Related in: MedlinePlus